Peptidomimetic agents from dextrorotatory amino acids as well as pharmaceutical agents that contain the latter for treatment of neurodegenerative diseases

ABSTRACT

A peptidomimetic agent from dextrorotatory amino acids includes vGek with Dval-gly-Dglu-Dlys as a central D-amino acid sequence, whereby gly is equal to D-glycine, which is equal to L-glycine. Pharmaceutical agents for use in the treatment of neurodegenerative diseases, in particular Alzheimer&#39;s disease, Parkinson&#39;s disease, Lewy Body dementia, Creutzfeldt-Jakob disease, as well as Huntington&#39;s Chorea disease, multi-system atrophy as well as disorders similar to these neurodegenerative diseases that contain at least one peptidomimetic agent from dextrorotatory amino acids are also included.

The invention relates to peptidomimetic agents from dextrorotatory aminoacids as well as pharmaceutical agents that contain the latter fortreatment of neurodegenerative diseases.

BACKGROUND OF THE INVENTION

In recent years, a number of peptides were found that were suitable forvarious pharmaceutical agent applications. In most cases, however,peptides cannot be taken orally, since they are often alreadymetabolized or in turn are very quickly excreted from the body beforereaching the site of action.

Peptidomimetic agents are peptide-like structures that can imitate orinhibit peptide actions as ligands. They are distinguished by a higherstability, by which the probability increases of reaching the site ofaction and exerting there an action as a pharmaceutical agent.

Aggregates that consist of incorrectly folded proteins very frequentlyoccur in the course of neurodegenerative diseases. In the case ofAlzheimer's disease, the extracellular deposits that are known as“plaques” from the literature, which mainly consist of β-amyloid,represent a main feature. The neurofibrillary bundles that occurintracellularly and mainly consist of hyperphosphorylated Tau proteinare another feature.

The investigation of β-amyloid plaque as well as the combatting of thesame has occupied a central role in the research of Alzheimer's diseasein recent years.

At this time, no treatment that acts on the causes of disease isavailable for treating Alzheimer's disease, Parkinson's disease, LewyBody dementia or other neurodegenerative diseases. The drug target thatis best known and best developed at this time for treating Alzheimer'sdisease deals with the β-amyloid-induced development of plaque.Alzheimer's disease affects 15 million humans in Europe and in theU.S.A. The incidence is 1-2% and increases to about 4% in the ninthdecade of life.

The deposits in the form of plaque mainly consist of amyloidogenicβ-amyloid. This amyloidogenic molecule species is formed by processingthe amyloid-precursor protein (APP) (Kang et al., Nature, 325 (6106):733-6, 1987; Goldgaber et al., J Neural Transm Suppl. 24: 23-28, 1987).APP, a transmembrane protein, whose physiological function is still notcompletely clarified (Selkoe, J Alzheimer's Dis., 3(1): 75-80, 2001), ispresent everywhere in the organism, but primarily in neuronal cells.

During and after the transport of the APP by the secretory pathway, theprotein can be cleaved in various ways (Goate et al., Nature, 349(6311): 704-6, 1991). The corresponding proteolytic activities are namedα-, β-, or γ-secretase. The activity of the α-secretase results in thecleavage of the soluble, extracellular portion of the protein (Esch etal., Science, 248 (4959): 1122-4, 1990). In this case, the transmembraneportion remains (non-amyloidogenic pathway). Another path of theprocessing contains the cleavage by β- and γ-secretase (amyloidogenicpathway). The cleavage by the β-secretase activity results in a somewhatsmaller secreted fragment (β-APPs) and a larger transmembrane portion(C-99). The C-99 fragment is then cleaved by the γ-secretase activitywithin the transmembrane portion. The amyloidogenic Aβ-peptides with 40or 42 amino acids (Aβ1-40, 1-42) result from this cleavage, which can becarried out primarily on two sites.

Aβ-1-42 has a strong tendency to form β-folded-sheet structures, whichare almost impervious to degradation by natural means. β-Folded-sheetstructures have a very strong tendency to form fibrils and ultimatelyturn into large aggregates.

In addition to the strong tendency toward aggregation, Aβ-1-42 isdistinguished in particular by a pronounced toxicity. Among otherthings, the presence of Aβ1-42 leads to an increase of oxidative stressas well as increased lipid peroxidation (Butterfield, D. A., Free RadicRes. 2002 December; 36 (12): 1307-13).

Aβ1-42 disrupts the functionality of the synapses and results inexcitotoxicity by a disruption of the cellular calcium balance. By thealready mentioned lipid peroxidation, it results in the production oftoxic oxidation products, which disrupt the functionality of ATPases,glutamate and glucose transporters (Mattson, M. P. and Chan S, L., CellCalcium. 2003 October-November; 34 (4-5): 385-97). The result is a lossof nerve cells by necrosis or apoptosis.

A number of in-vivo and in-vitro models, which impressively confirm therole of Aβ1-42 in connection with neurodegeneration, exist. Intransgenic mouse models, behavior deficits were also clearlyattributable to Aβ31-42.

The U.S. Pat. No. 5,985,242 describes retroinverse sequences thatconsist of D-amino acids, derived from the amino acid range 17-21 ofβ-amyloid. The molecules described there influence the aggregation ofβ-amyloid.

WO-03/082906 A discloses peptides that are derived from N-terminalsequences of the β-synuclein and exert protective action relative to theneurotoxicity of β-amyloid.

The direct prevention of the aggregation behavior of β-amyloid bysequences that are directly derived from β-amyloid protein, as proposedin U.S. Pat. No. 5,985,242, involves the risk, however, of additionalcomplications, since, as already known per se, small amyloid-likepeptides can be directly neurotoxic. However, the inhibition of theneurotoxicity exerted by already aggregated Aβ1-42 (i.e., withoutexplicit influence of the aggregation behavior) could even offer a greatadvantage. The peptides built up to form L-amino acids and described inWO 03/082906 A meet this requirement, but have a stability that is notquite optimum for use as pharmaceutical agents.

SUMMARY OF THE INVENTION

The object according to the invention was thus to find peptidomimeticagents that exert specific protection against amyloid-mediatedneurotoxicity, but have no similarities to the amino acid sequence inβ-amyloid regardless of the direction in which they are read.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an MTT test with the nuclear sequence ofvGEK.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, a peptidomimetic agent from dextrorotatoryamino acids is proposed, which is characterized in that it comprisesvGek with Dval-gly-Dglu-Dlys as a central D-amino acid sequence, wherebygly is equal to D-glycine, which is equal to L-glycine. Additionaladvantageous configurations of this peptidomimetic agent according tothe invention are disclosed according to subclaims.

Moreover, the invention comprises pharmaceutical agents for use in thetreatment of neurodegenerative diseases, in particular Alzheimer'sdisease, Parkinson's disease, Lewy Body dementia, Creutzfeldt-Jakobdisease, as well as Huntington's Chorea disease, multi-system atrophy aswell as disorders similar to these neurodegenerative diseases thatcontain at least one peptidomimetic agent from dextrorotatory aminoacids, which comprises vGek with Dval-gly-Dglu-Dlys as a central D-aminoacid sequence, whereby gly is equal to D-glycine, which is equal toL-glycine.

Other advantageous configurations of the pharmaceutical agent accordingto the invention are disclosed according to subclaims.

Furthermore, the invention relates to the use of the peptidomimeticagents according to the invention for the production of a pharmaceuticalagent for use in the treatment of neurodegenerative diseases, inparticular Alzheimer's disease, Parkinson's disease, Lewy Body dementia,Creutzfeldt-Jakob disease, as well as Huntington's Chorea disease,multi-system atrophy as well as disorders that are similar to theseneurodegenerative diseases.

An essential advantage of the peptidomimetic agents according to theinvention, which consist of D-amino acids as well as glycine, is thehigher stability compared to the degradation by various enzymes thatoccur in the body—in particular peptidases. For this reason, thestability of the nuclear sequence mimetic agent vGek was compared tothat of the corresponding retroinverse peptide KEGV according to WO03/082906 A, whereby the peptides together with a rat brain homogenatewere incubated for 15 minutes as well as for 1, 3 and 24 hours.

To this end, freshly isolated rat brain was homogenized in tris buffer(pH 7.6) and used immediately. In each case, a portion of a 1 mmolpeptide solution and four parts of the homogenate were incubated at 37°C. After the incubation, the decomposition of the enzymes was halted byadding 50 μl of 0.1 M HCl. Then, a denaturation of the proteins wascarried out with methanol (volume ratio: 1:4) at −20° C. for 60 minutes.Then, it was centrifuged for 20 minutes at 4° C. and 29,000 g.

Finally, the samples were concentrated under vacuum and analyzed bymeans of RP-HPLC (liquid chromatography, with UV/vis detector, detectionwavelength: 220 nm; solvent: (A) 0.1% TFA/water (A), 0.1% TFA in 80%ACN/water (B); column: Phenomenex Jupiter C18 (250×4 mm, 5 μm of 300 Å),flow rate: 1.2 ml/minute, gradients: 0 to 15% B after 30 minutes(KEGV_(OH)), 0% isocratic B for 10 minutes, then at 0 to 15% B after 30minutes (KEGV_(NH2)), 0 to 15% B after 20 minutes (vGek_(NH2))).

As an alternative, LC-MS (Applied Biosystems 140C Microgradient System;solvent: 0.1% HCOOH/water (A), 0.1% HCOOH in 80% acetonitrile/water (B);column: Alltech Hypersil C18 (150×2.1 mm, 5 μm of 100 Å), flow rate:0.15 ml/minute, gradient: 0 to 15% B after 20 minutes; detector:Finnigan TSQ-7000 Triple Quadrupole Mass Spectrometer, m/z Range;10-1000, scan time: 1.0 second) was also used.

It was finally confirmed that the peptide KEGV is comparatively quicklydegraded both in amidated form and also in non-amidated form. After aslittle as 15 minutes, only about 50% of the substance used is left, andafter one hour, complete KEGV can no longer be detected. The nuclearsequence of the mimetic agent vGek, however, has a significantly higherenzyme resistance: barely after 24 hours, vGek_(NH2) can be detected inthe test batch.

Sample T_(1/2) 15 Minutes 1 Hour 3 Hours 24 Hours KEGV_(OH) <15 14% 0% 0%  0% Minutes KEGV_(NH2) <15 22% 0%  0%  0% Minutes vGek_(NH2) 20-24 —— 70% 50% Hours

The respective half-lives (t_(1/2)) of the examined peptides can be readfrom the table. In columns 3 to 6, it is possible to read what portionof the amount of sample used is left after 15 minutes, and it was stilldetectable after 1, 3 and 24 hours. It is evident from this that underthe selected in vitro conditions, which represent an approximation onthe ratios in the brain, the stability of the peptidomimetic agent vGekaccording to the invention is around 2-3 orders of magnitude above thatof the corresponding peptide KEGV.

To examine the action of the peptidomimetic agent according to theinvention in a relevant biological system, injury tests were performedin cell cultures with Aβ-1-42 (β-amyloid). Differentiated SHSY-5Y cellswere used as cell culture systems.

The cells that were still not differentiated were cultivated in 96-wellplates at 37° C. in an atmosphere in DMEM/F12 (Cambrex) that contains 5%CO₂ until confluence is completed. For differentiation, the followingmedium composition was selected: 10 μmol of All-Trans-retinoic acid, 10%FBS, 4 mmol of L-glutamine, 200 units/ml of penicillin, 200 μg/ml ofstreptomycin, 0.5% DMSO in MEM non-essential amino acids (e.g.,Cambrex). After 8 days in the differentiation medium, 3×10⁵ cells/cm² inthe form of a monolayer were present.

Before the application, the test substances were irradiated for 10minutes in a form already dissolved in culture medium. This mediumcontained 2% FBS (fetal bovine serum) and was free of phenol red,retinoic acid and DMSO.

Aβ-1-42 was pre-aggregated before the addition for 24 hours at roomtemperature while being shaken lightly.

To damage the cells, the differentiation medium was drawn off andreplaced by new medium, which contained 2% FBS, 10 μmol of aggregatedAβ-1-42 as well as 50 μmol each of the peptide to be tested.

To determine the number of surviving cells, 40 μg of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium-bromide (MTT) wasadded per well and incubated for 3 hours at 37° C. Regarding theprinciple of the method: only living cells can turn yellow MTT intoblue-colored formazan crystals.

Then, the medium was carefully emptied, and the formazan crystals thatwere produced were dissolved with a DMSO/ethanol solution (4:1).Finally, the color intensity obtained was measured at 550 nm in an ELISAreader.

The properties of the nuclear sequence vGek were examined with an MTTtest on the survival rate of SHSY-5Y cells.

From this, it can be seen that the survival rate in the presence of 10μmol of pre-aggregated Aβ-1-42 drops to 56%. The third column, which isreferred to as vGek, shows that vGek at a concentration of 50 μmol hasno detectable influence on the survival of cells. Based on the extremerighthand column (vGek+Aβ 5:1), it can be shown that 50 μmol of thepeptidomimetic agent vGek cannot drop the survival rate despite thepresence of 10 μmol of Aβ-1-42. This means in toto that the nuclearsequence vGek in the concentration that is used is not toxic, but isable to almost completely prevent the damage of the SHSY-5Y cellsproduced by Aβ-1-42.

The chemical synthesis of a peptide from D-amino acids as well as alsothe production of the nuclear sequence vGek can be performed withconventional processes, for example with the Merrifield Solid PhaseSynthesis Technology (Merrifield, J. Am. Chem. Soc., 85: 2149-2154,1963; Kent et al., Synthetic Peptides in Biology and Medicine, 29 ff edsAlitalo et al., Elsevier Science Publishers 1985; Haug, J. D., PeptideSynthesis and the Protecting Group Strategy, American BiotechnologyLaboratory, 5 (1), 40-47, 1987). The processes for chemical peptidesynthesis also comprise the use of automatic peptide syntheses with useof commercially available protected amino acids, such as, for example,Biosearch (Model 9500 and 96001), Applied Biosystems Inc. (Model 430),Miligen (Model 9050), etc.

For the production of the peptidomimetic agents that are expanded aroundthe nuclear sequence, as disclosed according to claim 3, the usualderivatization is carried out at the C-terminal and/or N-terminal end ofthe nuclear sequence VgeK.

Thus, lysine and the amino-terminal end can be derivatized withsuccinate or other carboxylic acid anhydrides. These reactions have theeffect that the charge of the lysinyl radical is reversed. Othersuitable reagents for derivatization of alpha-amino-containing radicalscomprise imido-esters, such as methyl-bicolinimidate,pyridoxal-phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methyl-isourea, 2,4-pentanedione andtransaminase-catalyzed reactions with glyoxylate.

Free carboxyl-side groups (e.g., glutamyl) can be selectively modifiedby reaction with carbodimides (R′—N—C—N—R′), such as1-cyclohexyl-3-(2-morpholinyl (4-ethyl)) carbodimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide.

Other modifications include the hydroxylation of lysine, the methylationof the α-amino group of lysine and arginine, as well as the acetylationof the N-terminal amino group and the amidation of the C-terminalcarboxyl groups. Examples of such derivatizations are the formyl, acetyland propionyl amides as well as the methyl amides and dimethyl amides.All methods that are mentioned here for derivatization are familiar tothe peptide chemist.

In general, the compounds according to the invention are administered intherapeutically effective amounts in connection with pharmaceuticallyacceptable vehicles or solvents. Such vehicles comprise, for example,physiological common salt solutions, buffered common salt solutions,dextrose, water, glycerol, ethanol and combinations thereof. Therespective formulation is to be matched to the type of administration.

The composition can also contain, if necessary, varying amounts ofmoisture donors or emulsifiers or pH-buffering substances. Thepharmaceutical composition can be a liquid solution, a suspension, anemulsion, a tablet, a pill, a capsule, a retard formulation or a powder.The preparation can also be produced as a suppository with commonbinders and vehicles, such as triglycerides. Oral formulations cancontain standard vehicles, such as mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and othersin pharmaceutical purity. Various administration systems are known andcan be used to ensure the therapeutic application of the substancesaccording to the invention, such as, e.g., the encapsulation inliposomes, microparticles and microcapsules.

The form of administration is prepared in accordance with the processesthat are common in pharmaceutics, for an intravenous administration inhumans or other mammals. Typically, the compositions for intravenousadministration are present in the form of a sterile, isotonic, aqueousbuffer solution. If necessary, the preparation can also containsolubilizers and locally active anesthetics to alleviate the pain at theinjection site.

In general, the components are made available either separately or mixedin a metering unit, for example as a dry, freeze-dried powder oranhydrous concentrate in a hermetically sealed container, such as anampoule, on which the amount of the active pharmaceutical agent activeingredient is recorded.

If the dispensing form can be administered as an infusion, it can bedissolved in an infusion flask, which contains sterile water or saltsolution in pharmaceutical purity. If the preparation is to beadministered by injection, an ampoule with sterile water for injectionpurposes or common salt solution can be made available so that theindividual components can be mixed according to directions beforeadministration.

The therapeutic substances, which are described in this application, canbe formulated both in free form and as pharmaceutically compatible salt.Pharmaceutically compatible salts are those that were formed with freeamino groups, e.g., those that originate from hydrochloric acid oroxalic acid and those that are formed with free carboxyl groups, such asthose of sodium, potassium, ammonium, calcium, iron oxides,isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine,etc.

The amount of the pharmaceutical agent that is described in thisinvention must be effective for the treatment of the special disease orcondition; this depends on the type and the degree and is determined bystandardized clinical processes.

Thus, based on tests on rats, it was demonstrated that thepeptidomimetic agents according to the invention pass through theblood-brain barrier, although these are occasionally long-chain, i.e.,bulky molecules. The test was performed as follows:

Rats were injected with 2.94 mmol of tritiated vGek (vGek of specificactivity: 34.0 Ci/mmol). 10, 30 and 60 minutes after the injection, theanimals were anesthetized, and blood as well as a few organs wereremoved for examination and weighed. Depending on the organ, 50-250 mgof tissue was removed in a solvent and incubated at 60° C. for 2 hours.After cooling to room temperature, 200 μl of 30% H₂O₂ was added. Then,another incubation at 60° C. was carried out for 30 minutes. Finally,the radioactivity was determined with the aid of a scintillationmeasuring device.

Result:

The radioactivity reached its maximum in the brain after 30 minutes. Thehighest measured value in the brain was 1.33 pmol. It can be assumedfrom this that vGek could overcome the blood-brain barrier.

The exact dose, which must be used according to the invention, alsodepends on the type of administration and the degree of severity of thedisease or the disorder. This amount should be adapted with allowancefor the specific circumstances of the patient, based on the attendingphysician's assessment. Suitable dosage ranges for intravenousadministration are in general between 20-4,000 μg of the activecomponent per kg of body weight. Suitable dosages for intranasalapplications are in the range of between 0.01 mg per kg of body weightup to 1 mg per kg of body weight. Effective dosages for oralapplications are in the range of 1 mg to 1,000 mg per kg of body weightand day. The effective dosages are extrapolated from the dose-actioncurves, which are derived from in vitro models or animal model testsystems.

In summary, it can be stated that the peptidomimetic agents according tothe invention from dextrorotatory amino acids and glycine are stableagainst enzymatic destruction; this is produced by the dextrorotatoryform of the amino acids, whereby gly equals D-glycine, which is equal toL-glycine. An elevated stability in the metabolism and accordingly atarget-oriented action on the desired site of action can thus beproduced. The peptidomimetic agents according to the invention thatcontain pharmaceutical agents can inhibit at least the further build-upof the plaques that occur in neurodegenerative diseases and aretherefore relatively free of side effects, since the nuclear sequencevGek shows no toxic effects in the organism whatsoever.

1. A peptidomimetic agent from dextrorotatory D-amino acids comprising acentral nuclear sequence vGek with Dval-gly-Dglu-Dlys, whereby glyequals D-glycine, which is equal to L-glycine, wherein, the centralnuclear sequence is expanded on its N-terminal and/or C-terminal end byadditional dextrorotatory D-amino acids, and selected from the group:rervGek, vGekrer, ervGek, revGek, vGeker, and vGekre, whereby r=D-arginine, e =D-glutaminic acid, v =D-valine, G =D-glycine =L-glycine,k =D-lysine.
 2. A pharmaceutical agent for treatment of Alzheimer'sdisease comprising at least one of the peptidomimetic agents accordingto claim 1 as an active ingredient.
 3. A pharmaceutical agent accordingto claim 2, wherein the pharmaceutical agent is prepared for oraladministration.
 4. A pharmaceutical agent according to claim 2, whereinthe pharmaceutical agent is prepared for rectal administration.
 5. Apharmaceutical agent according to claim 2, wherein the pharmaceuticalagent is prepared for administration by inhalation.
 6. A pharmaceuticalagent according to claim 2, wherein the pharmaceutical agent is preparedfor transdermal administration.
 7. A pharmaceutical agent according toclaim 2, wherein the pharmaceutical agent is prepared for transmucosaladministration.
 8. A pharmaceutical agent according to claim 2, whereinthe pharmaceutical agent is prepared for administration with the aid ofimplants that contain active ingredients.
 9. A pharmaceutical agentaccording to claim 2, wherein the pharmaceutical agent is prepared forintracerebroventricular administration.
 10. A pharmaceutical agentaccording to claim 2, wherein the pharmaceutical agent is prepared foradministration by injection.
 11. A pharmaceutical agent according toclaim 2, wherein the pharmaceutical agent is prepared for transnasaladministration.
 12. A pharmaceutical agent according to claim 2, whereinthe pharmaceutical agent is prepared for administration by infusion.